The field of the invention relates to in vitro synthesis of proteins. In particular, the field of the invention relates to one-pot systems for incorporating non-standard amino acids into cell-free synthesized proteins.
Cell-free protein synthesis (CFPS) using extracts from prokaryotic source strains such as E. coli has undergone a transformational shift from an exploratory platform used in the discovery of the genetic code to a present-day, high-yielding protein production platform [1]. This shift is fueled by the open nature of this system, allowing for rapid combination, supplementation, and optimization of the physiochemical environment for increasing protein yields and batch reaction duration [2, 3]. Now, cell-free systems are seen as a complement to in vivo protein expression and can be used as both a prototyping platform due to its simplicity, easiness, and modular design for protein expression [4-6] as well as a large-scale production platform for difficult to express proteins in vivo [7]. The transition from exploratory platform to high-yielding protein production platform has come about, at least in part, by complex strain engineering to stabilize biological substrates in the cell-free reaction mixtures [8, 9]. These genetic modifications targeted the deletion of proteins known to affect the stability of DNA [10], mRNA [8, 11], protein [12], energy [13], and amino acids [14, 15] in the cell-free reaction. In addition to strain engineering efforts, activation of multiple biological pathways [16], decreases in cost [17], and improved understanding of reaction contents makes CFPS an attractive platform for the production of new kinds of high-value proteins.
One area of great interest for the application of cell-free systems is the production of modified proteins containing non-standard amino acids. Incorporating non-standard amino acids or unnatural amino acids (NSAAs) allows for the production of proteins with novel structures and functions that are difficult or impossible to create using the 20 canonical amino acids [18, 19]. Recently, cell-free protein synthesis (CFPS) systems have been employed to increase yields of proteins bearing NSAAs [20, 21], achieve direct protein-protein conjugation [22], explore drug discovery [23], and enhance enzyme activity [24, 25].
Typically, NSAA incorporation systems use amber suppression technology to insert NSAAs into proteins, a method by which an in-frame amber (TAG) stop codon is utilized as a sense codon for assigning NSAAs [26, 27]. Amber suppression technology, however, has limited efficiency for NSAA incorporation because of the presence of release factor 1 (RF1). RF1 naturally binds the amber stop codon (TAG) [28] and prematurely terminates protein translation. Methods to counteract this competitive termination of the TAG stop codon include increasing the addition of competing tRNA [21], tagging and purifying out RF1 [29], release factor engineering [30], and genomically recoding strains to remove RF1 and reassigning all occurrences to the synonymous TAA codon [31]. High-yield protein production with multiple-site incorporation of NSAAs still remains a critical challenge.
In addition, cell-free protein synthesis systems may involve coupled transcription and translation of a target protein. Such systems generally utilize a transcription template for synthesis of mRNA encoding the target protein. The transcription template typically will include a strong heterologous promoter for a heterologous RNA polymerase, such as the promoter for the bacteriophage T7 RNA polymerase. Such systems either need to be supplemented exogenously with the heterologous RNA polymerase in order to transcribe the target protein mRNA or the source strain that provides the cell extract for the cell-free protein synthesis system must be engineered to express the heterologous RNA polymerase. Unfortunately, source strains that have been engineered to express a heterologous RNA polymerase may not express the heterologous RNA polymerase at sufficient levels and/or native proteinases of the source strain may recognize and cleave the heterologous RNA polymerase at cryptic cleavage sites, rendering the heterologous RNA polymerase non-functional. In addition, an extract from a source strain that has been engineered to express a heterologous RNA polymerase otherwise may not function as well when utilized in cell-free protein synthesis as an extract from the native source strain. Therefore, optimized strains that express heterologous RNA polymerases, protein production platforms, and methods for producing modified proteins containing NSAAs in high yields are needed.